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Volume 10, lssue
I
77
VIBR IO P A R A HAEMOL YTZCUS A N D R E L A T E D H A L O P H IL I C
VI BRI OS
Authors: Sam W. Joseph
Rita R. Colwell
Department of Microbiology
University of Maryland
College Park, Maryland
James B. Kaper
Center for Vaccine Development
University of Maryland Medical School
Baltimore, Maryland
Referee: A .
von Graevenitz
Institute of Medical Microbiology
University of
Zurich.
Zurich. Switzerland
I.
I N T R O D U C T I O N ( H I S T O R Y )
Th irty years have passed sinc e the first isolation of Vibriopatahaemolyricuus.Volumes
of literature have been w ritten a bo ut this organism a nd it has become recognized as a
ma jor cau se of food p oisoning in area s of the world where seafood is a majo r staple of the
diet. Th e history of V parahaemolyricus traces back to O ctobe r 20a nd 21,1950, when an
out bre ak of food poisoning occurred in the southern su bur bs of Osaka, Jap an . Of the
272
patien ts who suffered acute gastroenteritis,
20
died. The sym ptom s of the gastroenteritis
included acute ab dom inal pain, vomiting, and diarrhea, with watery and , in som e cases,
bloody stools. Th e food suspected to ha ve caused the foo d poisoning was a small, half-
dried sardine, Engradis aponica Ho uttu yn, called "shirasu" in Japanese. Th e sardine is
boiled in salt water and eaten when partially dried.'
Based on his expertise derived while assigned at Maym yo, B urma in 1944 as an arm y
surgeon sublieutenant,
T.
Fujino and his co-workers at the Research Insti tute for
Microbial Diseases (Osaka University) carried
o u t
the bacteriological investigation of
specimens from the intestinal tracts
of
the victims and the shirasu suspected to be the
source of the organism.*-' During his Burma tour of duty, he diagnosed two cases of
bubonic plague, by guinea pig inoculation, ob taining a pure c ultu re of plague bacilli from
spleen and ascites. Thus, Fujino injected filtrates of the homogenized shirasu via the
intraperitoneal route into guinea pigs, the objective being to exclude chemical poisons
and pleuro pneum onia-like, filterable bacteria as causative agents. Pu ru len t peritonitis
was induced in a guinea pig. Th e saline hom ogenate of shirasu w as centrifuged and the
supernatant was inoculated into various culture media and incubated at 37C under
aerobic and anaerobic conditions. Salmonella. Shigella. Clostridium, and
Proteus
were
not found. However, many colonies
of
what was thought to be
hctobac i l lus
and
Sruphylococcus were observed, along with many Gram-negative rods. The Gram-
negative rods formed whitish, opaque colonies and appeared microscopically to be
similar to Escherichia. In a smear of a single colony, Fujino observed a few Gram-
negative bacteria w ith swollen edges am ong the num erous G ram-negative rods. Attempts
to separate the two kinds of Gram-n egative bacteria by transfer to fresh agar plates were
unsuccessful. Thus, hypothesizing that one of the two G ram-neg ative bacterial strains
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was a pathogen and would inhibit growth of the other strain in vivo and grow more
rapidly, F ujino an d his co-workers attempted sep ara tion of the two by anim al passage. A
suspension of colonies co ntai nin g the tw o kinds of Gram -negative ba cteria was injected
intraperitoneally into mice. Several h ou rs later, when sym ptom s of illness appeared in
the mice, samples of ascitic fluid were tak en an d inoculated into oth er mice. The anim al
passage was repeated once again, and after 2% o
3
hr, sym ptom s appeared in the mice.
The asc itic fluid f ro m the last set of mice to be passaged was inoculated onto blood aga r
plates and the plates were incubated at 37C for 10 hr. Two types of colonies were
observed on the plates: a nonhemolytic, slender, Gram-negative rod, which on
subsequen t testing in pur e culture produced acid an d gas in glucose an d did not hydrolyze
gelatin, and a hemolytic, fat rod demonstrating bipolar staining. The latter isolated
produced acid - ut not gas rom glucose, liquified gelatin, an d proved pathogenic
for mice. The gas-producing strain was identified as
Proreus morganii, but the
anaerogen ic strain could not
be
identified a t tha t time. T he history of this series ofe ven ts
in the isolation of V .parahaemolytica is nicely detailed by F ujino' an d by Miw atani and
Takeda.6
The anaerogenic, unidentified bacterium of Fu jino was tested fu rther and found to be
actively motile by means of a single, polar flagellum, resembling
Vibrio cholerae.
However, it did no t react with
V. holerae
ant iserum and the long axis
of
the bacterium
was not curved. B ased o n these features an d o n its bipola r staining, the new species was
named Pasteurella parahuem olyticus [sic], n. sp.'
Fu jino 2 subsequently isolated the sam e bacterium fro m the intestinal contents of a
victim of foo d poisoning, an d Fujino et al.' rep orte d the discovery of the new pathogen a t
the 25th Annual Me eting of the Japan ese Association fo r Infectious Disease in
195
1.
An
extensive, detailed description of Pasreurella parahaemolytica first appeared in
Jap an ese 2 and later in English.'
In
the fall of
1955 I.
Takikawa,' from the Nation al Yokoh am a Hospital, visited the
laboratory of Dr. Fuj ino. An outbreak o ffoo d poisoning had occurred at the Yokoham a
Hospital, involving
I20
patients, with no deaths. A Gram-negative rod with a single,
polar flagellum was isolated on 4% salt ag ar used to isolate Staphylococcus. The culture
appeared to be similar to Pasreurella parah aem olytica. but was halophilic, the salt-
requiring na ture of
P. parahaemolyrica
not having been recognized previously. In the
hospital inciden t, the foo d implicated was brine cuc um ber (pickles) served to the patients
and it was speculated tha t the causative agent was a halophilic orga nism from mackerel
which had contaminated the cucumbers.7.*
The description of Pasreurella parahuemolyticus provided by Fujino et a].' was as
follows: "A rod-shaped organism. 1 to 3
p
in length, w ith rou nd ed ends, which is slightly
pleomorphic
on
blood agar. It shows a tendency toward bipolar staining, and when
stained with weak Giemsa solution for 15 min, gives a clear bipolar picture. Direct smears
from anim al blood show th at it has a thin capsule. It
is
monotrichal.
A
very few organism s
can be seen to mo ve like
Vibrio comm a.
When an 18-hr blood agar culture is examined
und er d ark field i l lumination o ne o r two bacill i per field c an be seen to move. Electron
microscopy shows th at each bacillus has a single flagellum. Mo veme nt c an also be seen in
direct pr epa ratio ns from mouse ascites. It liquifies gelatin, an d show s a hemolytic ring in
blood agar. It grows aerobically on plain ag ar f orm ing white, op aqu e colonies."
Ta kik aw a7 rep orte d tha t his isolate was closely related in its characteristics to the
organisms described by F ujin o et al.,3 but he concluded tha t it sho uld be considered a
member of the genus Pseudomonas." i.e., Pseudom onas enreritis. Thereafter , in Ja pan ,
the organism was referred to
as
the so-called "pathogenic halophilic bacterium".
M iyam oto et aL9 stated tha t the organism shou ld be placed in the genus Aeromonas
because of its fer me ntat ive utilization of glucose, and proposed a new genus,
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Oceanomonas, for the halophilic, fermen tative bacteria. Th us M iyamoto et aL9proposed
that the clinical isolates of Fujino et aL 3 and Takikaw a" should be classified together
with
their own strains isolated from feces and seawater in the new genus as
0
parahaemolytica,
0.
enteritidis. and
0.
alginolytica.
Sa ka zak i and co-workers" first placed the strains into the genus Vibrio after
examination of
1072
such strains. Sub sequently,
K
parahaemolyticus was divided into
two biotypes, which Zen-Yoji et a1.I' concluded should
be
separated into two species.
This was propose d by Sakazaki" in 1968, wh o designated biotype 2 as
V.
alginolyticus.
The main differences between the biotypes were acetoin production, sucrose
fermentation, growth in 10% NaCI, and swarm ing on agar plates containing
2
to 7%
NaCI.
V.
parahoemolyticus is negative for these characteristics and
K
alginolyticus is
positive.
The 8th edition of
Bergey
s
Manual
of
Determinative Bacteriology"
designated
V.
alginolyticus as biotype 2 of
V.
parahoemolyticus. but the Subcommittee on the
Taxonomy of Vibrios of the International Committee on Systematic Bacteriology
recognized the sep aration of these two phen a a s distinct species." A third biotype was
discovered re~ently.'~' ' ' reviously regarded as "lactose positive" vibrios, the nomen-
clature Vibrio vuln ficus was proposed.18
Detailed reviews of the history of the taxonomy of
K
purahaemolyticus have been
provided by Colwell, ' Cabassi and M on, l9 Miwatani and Take da,6and C hatterjee.20 n
recent y ears, pro posa ls have been made t o reassign this species from the genus Vibrio to
the family Brucellaceae by Chatterjee" an d to the genus Beneckea by Baumann et a1."
The definition of the genus
V i b r i ~ ' ' , ~ ~ncludes straight rods and embraces
V.
parahuemolyticus.
since it produces a p ola r flagellum when grown in broth, even tho ugh
it
m ay be peritrichously flagellated when gro wn on a ga r medium (vide infru). ,While V.
parahuemolyticus may require slightly higher concentrations of Na' than V. cholerae.
which re quires only trace concen trations,
V .
cholerae demonstrates a salinity op timum of
approximately 1 NaCl as opposed to 2 to 3% NaCl for V. parahaemolyticus,"
differences not suitable for generic separation. In vitro D N A / DN A h ybridization d ata
also show that
V.
parahuemolyticus
is
more related genetically to
K
cholerae than to
other Beneckea species." Recent pub lications have demo nstrate d a concensu s on
retaining this species in the genus V i b r i ~ . ~ '
11.
TAXONOMY
A. Morphological, Cultural, and Biochemical Characteristics of Y.parahoemolyticus
I . Morphology
Viewed with the microscope, V. parahaemolyticus appe ars as a straight, sometimes
curved rod with rounded ends, pleomorphic, and usually occurring as single cells but
occasionally in chains. Undulating filaments and spheroplasts are often present."
V.
parahuemolyticus is Gram-negative and occasionally a concentration of the G ra m stain
ap pe ar s at the p olar extremities of y ou ng cells in the process of dividing.I9 Th e
flagellation of this organism has been extensively studied and mo st workers agree that
V.
parahaemolyticus
possesses a single, polar, sheathed flagellum when grown in liquid
medium and, in addition, unsheathed, peritrichous flagella when grown on solid
media.22.26-29
K i
mura et aL3' reported that formation of peritrichous, but not polar
flagella, was inhibited
in
media of pH 8.5 and higher.
2.
Physiology and Resistance
to
Antibacterials
V parahuemolyticus grows within a temperature range typical of mesophiles, with a
minimum growth temperature of 9 to
IO'C,
a m aximum growth tempera ture of
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approximately 4 4 O
C,
an d a n opti m um between 35 and 37' C."-3J In teracting effects of
pH , temperature, an d salt concen tration can esult in slight alteration in the temp eratu re
limit on g rowth . B e ~ c h a t ' ~eported moderate grow th occurred
at
5C when the medium
was in the alkaline pH range. T he pH r ange
of
growth
of
V.
paruhaernolyricus
is fairly
wide. Initial pH of media permitting growth ranges from pH 5 to
1
I, with an optimum
between pH 7.5 and
8.0.1'*34J5
T he ability of
V. arahoemolyticus
to grow at high pH has
been exploited in the development of selective isolation media
(vide infru).
V parahuemolyticus
is a moderate halophile for which salt requirement and salt
tolerance have been extensively studied. Gro wth occurs at NaC l conce ntra tion s
of
0. I M
to approxim ately 1.2 M i th an opt imum sal t concentrat ion for growth of about 0 .5 M
NaCI.1'*3'-39Distilled water inactivates
V. aruhaemolyricus,
with a
90%
reduction
of
viable cells within 1 t o 4 min." Ot he r cations, e.g., Li' an d K', can produce a sparing
effect on th e specific requirements fo r sodium, but th e minimal, essential requirement
for
sodium is appro xim ately 0.003
to
0.007
M
Na', wh en the cells ar e grow n in synth etic
media."*41 When nonm etabolizable LiCl, sucrose, an d ethylene chloride ar e used to
replace sodium ion in a synthetic medium, each is capable
of
providing osmotic
regulation. In ad dition t o this requirement, sod ium ion ap pea rs to be necessary for
protein synthesis, which is inhibited in sodium ion-omitted medium containing osmotic
agents."*'"
In a s tudy of a ma r ine pseudomonad D rapeau and M a c L ~ o d " ~ound that washed
cells, when incubated w ith l'C-a-aminoisobutyric acid, were ab le t o accum ulate this
analogue inside the cell, with ou t metabolizing
it,
an d required the presence of Na' in the
medium.
K',
Rb', NH4', Li', an d sucro se could not su bs titu te fo r Na' in the tra ns po rt
process
.
In similar fashion Sa ka i and Sakai'"found tha t a gr ou p of marin e bacteria, which they
term T H (terrestrial halophilic) type (and include V.pmahoemolyricus) is similar to their
MH mar ine halop hilic) type w hich is described a s follows:
M
H-type ba cteria seemingly
require
Mg
for the oxid ation of substrates, however, Na' only is able to play
the ab ov e physiological roles. Na'. in fact, prevents th e lysis of cells an d accelerates
cytochrome oxidase, the electron tran spo rt chain, and A T P form ation of oxidative
phosphory lation. Na* also functions in the Na', K'dependent active tran spo rt of
nutrien ts in to the cells. K'is accesso nly needed only in the presence of Na'. Con sequ ently
MH -type indispensably needs Na' as the sole cation of su pp or tin g growth. This type
belongs t o psychrophilic bacteria because it lacks grow th capacity a t 37" C." Th e
exceptions which sep arate the T H type from th e M H type are described as follows: "The
Na' requirement is less than that of MH -type fo r the prevention of lysis, the oxid ation of
substrates, the electron tran spo rt system, the cytoch rom e oxida se, an d growth. T his type
is able to grow well a t 37C an d thereby belongs to mesophilic bacteria." Th e combined
effects of water activity, solute, and temp erature o n th e grow th of V .porohaemolyticus
have been studied with t he limiting water activity for g rowth d epend ing up on th e solute
employed.
An interesting physiological feature of V.
parahuemolyricus
is its extremely short
g en eratio n tim e. K a t ~ h ' ~eported a generation time
as
shor t as 8 to 9 min, althoug h 10 to
12 min is mo re com mo nly observed. UlitzurU studied 30 strains of
V.
parahoemolyticus
and observed two major groups, based on generation time. One group
of
strains
dem onstrated a short generation time, ix . , 12 to 14 min, and a second grou p, a longer
time of
20 to
25 min.
V. parahoemolyticus,
like other vibrios, is a facultative anaerobe, possessing both
respiratory an d fe rm enta tive metabolism.
I t
produces a catalase and cytochrorne
oxidase. It is sensitive to the vibriostatic agent
O /
129 (2,
4
diamino-6, 7-diisopropyl
pteridine) and is, in general, sensitive to ch loram phen icol, gentamicin, kan amy cin,
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nitrofurantoin, tetracyline, doxycyline, and streptomycin an d is resistant to ampicillin,
carbenicillin, clindamycin, colistin, erythromycin, and penicillin.'
J ~ ~ ~ ~ - ~ ~
para-
haemolyticus
and
V.
alginolyticus
from hum an and environmental sources, examined by
the minimal inhibitory concentration (MIC) method, are uniformly susceptible to
chloramphenicol an d tetracycline within attai nab le serum levels. Most strains are
resistant to ampicillin and exhibit p-lactam ase activity, which accou nts f o r this
resistance. Occ asional multiresistance is noted, bu t thus fa r plasmid-linked d ru g
resistance has not been shown. Susceptibility to gentamicin can be demonstrated with
agar diffusion, but examination for MIC in brain heart infusion broth, containing 2%
NaCl, yields inconclusive results because of diminished gentamicin activity.*8
3. Biochemical and Nutritional Characteristics
Several investigators have provided numerous biochemical and physiological
characteristics of
V.
parahaemolyticus.
1 * r 9 - 5 '
V.
parahaemolyticus ferments glucose without the production of gas and does not
produce acetoin, i.e., it is Voges-Proskauer negative, ferments galactose, levulose,
maltose, mannitol, mannose, ribose, and trehalose, but does not ferment adonitol,
dulcitol, erythritol, inositol. lactose, melizitose, raffinose, rhamnose, salicin, sorbose,
sorbitol, sucrose, and xylose. Strain variation can be observed in the fermentation of
arabinose, cellobiose, and melibiose.
V. parahaemolyticus is positive for production of indole, and possesses lysine and
ornithine decarboxylase, reduces nitrates to nitrites, an d de grades gelatin, chitin, starch,
casein, and lecithin. It is usually negative for arginine dihydrolase activity, hydrogen
sulfide production (vide infru), urease, phenylalanine deam inase, and luminescence. An
im po rta nt , but variable, characteristic is hemolysis of blood, kno wn as the K anagawa
phenomenon (KP), measured by using a high salt-containing medium (vide infra).
In
general, strains
of V .
parahaemolyticus utilize th e following co mp ou nds as sole
sources of carbon: D-glUCOnate, acetate, citrate, propionate, DL-malate, pyruvate,
fumarate, lactate, succinate, ketoglutarate, ethanol, propanol, L-serine, L-leucine,
L-
glutamate, L-arginine, L-proline, L-tyrosine, L-alanine, L-arginine, and L-histidine.
I t
cannot utilize phenol, catechol, malonate, oxalate, glutarate, tartrate, p-hydroxyben-
zoate, butanol, benzoate, p al an in e, L-ornithine, L-citrulline, or spermine."J4Jo
4.
Deoxyribonucleic acid
(DNA)
Base Composition and Nucleic Acid Hom ology
The composition of chromosomal DNA
of
strains
of V.
parahaemolyticus is in the
range 44 o 46% guan ine plus cytosine (G
+C).
D N A /D N A reassociation studies have
been performed by several investigators an d intraspecific values ar e usually >90% (at 60
to 63C ).24Jz-54nterspecific values between
V .
parahaemolyticus and V cholerae range
from 16 to 56% (at 60 C) w ith mo st values falling between 20 to
30 .52Js
alginolyticus
shares
60
o 70% homology with K parahaemolyticus and strains
of
the "lactose positive"
vibrios share 40 to 50 homology with V parahaemolyticus and V. a l g i n o l y t i c u ~ . ~ ~,
parahuemolyticus reveals only a low amount of homology with other marine vibrios,
specifically,
V.
anpi l larum. Reassociation values between these tw o species ar e in the
range of 20 to 30% with the mem brane filter whereas values of only 4 to
5
homology were observed using the S endonuclease assay, a m ore stringent method for
estimation of DNA homology.56
a PIasmids
Extrachromosomal elements of
V .
parahuemolyticus are evident but no specific
function has been assigned to them. Guerry and Colwel15' fo un d that 40 f 12s train sfrom
human and e nvironme ntal sources had multiple plasmid species of cryptic function.
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Table 1
MINIMAL CHARACTERS
FOR
DENTIFICATION OF
V .
PARA
H A
MOL YTICUS
Sign
Gram-negative. asporogenous rod
lndophenol oxidase
Glucose. acid under a seal
of
petroleum
Glucose.
gas
D-rnannitol. acid
Sucrose. acid
Acctylmethylcarbinol
Hydrogen sulfide, black butt
L-lysinc d ccarbo xylax
L-arginine dihydrolase
L-omithine dccarboxylase
Growth in 1 tryptone broth
Growth in
1 tryptone
broth with
8
NaCl
Growth in 1 tryptone broth with IO?6 NaCl
Growth at
42OC
From
Hugh.
R. and Sakazaki, R.. J . Con/: Publ. Healih
Lab.
Direct..
30,
133,
1972.
With permission.
5.
Minima l Characteristics f o r Identijication
of
V. parahaem olyticus and Biochemical
Variation
A
list of m inimum characteristics for identification of V. parahaemolyticus has been
repor ted by Hugh and Sakazaki and C0lwe11~~Table 1). A comparison of
characteristics fo r
V.
parahaemolyticus and related species and genera is provided in
Table 2. Individual characteristics sho uld not be overem phasized f or identification, since
stra in variability is co mm on. Conversely, even if a strain , particularly a n environmen tal
isolate, fulfil ls all cri teria, fu rther biochemical cha racteriz ation, even D N A / DN A
hybridizations, sometime m ay be required for c om plete separation an d identification.
A
more complete characterization of V. parahaernolyticus, beyond a minimum key
chara cter analysis,
will
occasionally distinguish an isolate from similar, but as yet
incompletely characterized, marine bacteria.
Exceptions
to
the list of minimal characterist ics shou ld be noted. F or example, sucrose
fermentation is a primary differential characteristic an d
is
th e basic criterion employed in
most of the isolation methods recommended for
V .
parahaemolyticus (vide infra).
Nevertheless, man y sucrose positive strain s have been r ep orte d. ColwelldO oun d 6% of
the strains of V. parahaemolyticus examined to be sucrose posit ive. Joseph and
G ilm our 6 also re ported several such s train s. K am pel ma che r et a1.62 isolated sucrose
positive
V. parahaemolyticus
from mussels and F ujin o et al. fou nd
2% of
their marine
isolates to be sucrose positive. Of
1484
strai ns of this species isolated fro m British coastal
waters,
6.9%
was
sucrose positive. Success in dete ctin g- acid produced du rin g
fer me ntat ion of sucrose by
V.
parahaemolyticus can vary , accord ing to the medium and
concentrat ion of sucrose used. B aross@ recom mend ed the use of
OF
basal medium ,6s
with 0.5% final conc entration of added sucrose. Se pa ratio n of V. parahaemo lyticus and
V . alginolyticus
must be based on several criteria, such
as
acetoin prod uction, grow th in
10% NaC1, swarming on agar plates, and methyl red reaction, in addition to sucrose
fermentation.
Lysine and ornithine decarboxylases are usually present in V. parahaem olyticus, but
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ma ny exceptions will be observed. Fujin o et al. repo rted
4
to
5
of the s train s tested to
be negative for ornithine a n d /
or
lysine decarboxylase. The percentage of ornithine
positive strains isolated fro m sam ples collected in Tok yo has been rep orted to be as low
as 78%.
Production of hyd rogen sulfide should be tested by reading the reaction occurring in
the butt of a tube of Kliglers Iron agar (KIA) or triple sugar iron aga r
(TSI).
Sakazak i et
al. reported that non e of 1702 strains examined were positive for H2Susing TSI or S I M
media. ColwellW fou nd nearly all of the stra ins of
V. cholerae
and V .
parahaemolyricus
examined to be
H2S,
using more sensitive methods for detection of
HIS
production.
T wedt and c o - ~ o r k e r s ~ou nd nearly all of their cultures to be positive using SIM and
lead acetate ag ar (Difco). Indeed, hydroge n sulfide pro duc tion o n Russells Triple sugar
agar, but not on
TSI, h a s
been suggested as a n aid in the identification of this species.67
Two o ther cha racteristics, althou gh no t included in the set of minimal characteristics
listed by H ugh an d Sakazaki,* are noteworthy. Ind ole pro duc tion is usually positive, bu t
indole negative strain s have been implicated in several outbr eak s of foo d poisoning in
Tokyo.66 V. parahaemolyticus is usually considered to be urease but
several investigators have reported other findings. Colwel16 found that 97% of
V.
parahaemolyricus stra ins tested was urease + and Ch itu et a1.,68who examined 8 strains
of V. parahaemolyricus isolated from salted herring and roe, found 6 to be urease
posit ive. Sakazaki et al . reported 4% of the stra ins tested to be urease positive and
Kaper et al. (in prep ara tion ) reported 13 urease positive strain s of 19 isolated from
shrimp. The isolation of urease positive strains from human infections is further
substant iated by the reports of Huq et al.69 and Lam and Y ~ o . ~
B. Other Biochemical Studies
The cell envelope of V. parahaemolyticus has been characterized by De neke and
Colwell and T am ur a et al . ,72*73nd has been shown to possess poly p-hydroxybutyric
acid and a highly branched a- D glu ca n. Fa tty acid com position was examined by
Rietschel et al . , Ol iver and C~lwel l ,~n d B euchat an d W ~ r t h i n g t o n , ~ ~nd the
predominant fatty acids were C12, C14, C16:l , C16, and C18:l . Using high pressure
liquid chr om otogr aph y, Me11 et al.,? also fo und C 14 :l, C13, CI S, C 17, C18, C19, and C 21.
Sup erox ide dismutase
(SOD)
nd catalase levels in
V. parahaemolyricus
were examined
by Daily et al.,* who found only one detectable SOD nzyme in most of the strains
tested, as compared with the three
SOD
enzymes of
E.
coli.
A c-type c ytoch rom e, with
the capacity to bind carbo n mo nox ide, was repo rted for the soluble fraction of cell-free
extracts of
V.
parahaemolyricus by Collins and Knowles (unpublished observations,
quo ted by West et al.. Th e function of this cyto ch rom e is un kn ow n but evidence is
accumulating that such cytochromes are associated with organisms having complex,
branched respiratory Oth er enzymes of
V. parahaemolyricus
which hav e been
studied include phospholipase
A ,
lysophospholipase and
glycerophosphorylcholine
diesterase, lysophospholipase, lecithinase,82aspar tok inase , amylase , ge la t ina~e,~
and acid and alkaline phosphatases.86
111. I S O LA T I O N A N D E N U M E R A T I O N OF V. P A W H A E M O L Y T I C U S
V . parahaemolyticus has captured the interest of microbiologists in many fields,
especially medical, fo od , and env iron m enta l microbiologists. As a result, a variety of
me thod s have been developed specifically
for
the isolat ion, en richment , and enum erat ion
of
V.parahaem~ly~icus.~~*-~
ost of the me tho ds involve direct plating of samples on
an aga r medium, o r initial enrichment in broth, followed by s t reaking and isolat ion on a n
agar medium. Direct plating is sufficient, in most cases, for fecal specimens, but food
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samples and samples collected from th e environment frequ ently, if not always, require
enrichment.
A
few procedures require special incubation of media at elevated
temperatures
(42
to 43C)
o r
under anaerobic conditions to provide additional
selectivity.
Typical colonies of V.
parahaemolyticus
are picked and tested using a set
of
biochem ical, physiological, an d sero logical tests. If imme diate processing of a sample is
not possible, samples can be frozen, but the risk of stress and death of cells must be
recognized. Samples also can be transferred to a tran spo rt medium , such as Cary-Blair
medium"
or
that described by LeClair et al.9'
A. Enrichment Broth
Initial contributions to methodology f or isolation and en ume ration of V.parahaemo-
lyricus
were m ade by Japa ne se investigators who developed selective media employing
agents or conditions such as Teepol (a neutral detergent), bile salts, dyes, high salt
concentrations, and alkaline pH. Glucose-salt-Teepol broth (GSTB), devised for
isolation
of V
parahaemolyticus, contains methyl violet and Teepol and is adjusted to
pH 9.4.93 GS TB has been m odified subsequen tly by substitution of lauryl sulfate f o r
T e e p 0 1 . ~ ~rabinose-ethyl violet broth" con tains ethyl violet an d is adjusted to pH
8.6.
A
collaborative study reported by Petersenm 3 described Horie bro th
as
yielding MPN
values ten times greater tha n GSTB . This medium was modified by Kape r et by
substitution of galactose for arabinose. Other enrichment broths proposed for V.
parahuemolyticus
include the SWYE medium of K ane ko a nd Colwe1I9' an d salt-colistin
broth (SCB) of Sakazaki." A salt-polymyxin B broth (SPB) was proposed by
Ka mp elma che r et al.," an d a mo re recent version of
SPB,
sed by Sakazaki (personal
comm unication), contains polymyxin B (500 pg/m l), salt
(2%
NaCl), and nu trient broth.
A
survey of several broths proposed for V.parahaemolyticus by Naka nishi an d MuraseIoo
showed that SPB provided the best recovery when raw fish was examined for the
organism.
A
simple medium, containing Teepol and
3%
NaCl in a pho sph ate buffer, was reported
by Ch un et a1.'" an d Teep ol has also been employed in a wa ter blue-alizarin yellow
b r ~ t h . ~ ' r i s t e n ~ e n ' ~ ~ * ' ~ 'tilized a meat broth amended with 7% NaCl and
0.3%
alkyl
benzene sulphonate, to which starch and chitin had also been added. Trypticase Soy
bro th (TSB), amen ded with 7%NaCI, has been used by Vand erzant and N ickelsonmas an
enrichment, with alkaline-saline peptone wateriM
as
a second enrichment after
incubation for 8 to 12 hr. Sakazak i" has used the tellurite-bile salt br oth of Mon surIo5 or
secondary enrichment. Bismuth sulphite phenol red (B SP R) bro th, containing sucrose,
NaCI, manni tol, and bismuth sulphite, was employed by Thompson and T r e n h ~ l m " ~or
isolation of V. parahuemolyticus.
A
recent study of
V.
parahaemolyticus in Dutch
mussels'07 included a comp arison of five enrichment broths. Th e conclusion was tha t
enrichment in the 5% NaCl meat broth
of
Kam pelmach er et incubated at 37C
provided the best recovery of
V
parahaemolyticus.
B. Plating Media
A wide variety of plating media, m any of which were originally developed fo r isolation
of
V. cholerae,
has been employed fo r recovery of V.
parahuemolyticus.
Undoubtedly,
the m ost comm only employed a gar m edium is the thiosulfate citrate bile salts sucrose
(TCB S) aga r of Kobaya shi et al.''' This medium inh ibits mo st of the othe r bacterial
species comp rising the fecal flora primarily because of the presence of bile salts, sodium
citrate, and the highly alkaline pH of 8.6. Good differentiation of Vibrio species is
provided by the sucrose fermentation reaction.
V. cholerae
and
V alginolyticus
ferment
sucrose, producing yellow colonies, indicated by color change in the brom thymol blue
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and thymol blue included in the medium. V. parahaemolyticus, generally, is not a
sucrose-fermenting organism on TC BS agar, a nd the refore prod uces a bluish or blue-
green colony. Although TCBS
is
a medium useful for isolation of
V.
cholerae
and
V
parahaemolyticus, the selectivity of the m edium is suc h tha t it may not always suppress
growth of other organisms, such as Proteus spp., Aeromonas spp.,
or
Staphylococcus
spp. Furth erm ore, differentiation am ong species of pa thoge nic and other, as yet poorly
characterized, species of
Vibrio
foun d in the aq uat ic environ me nt is not always achieved
and additional tests, comprising a follow-up screening, are required. Modifications of
TC BS have been proposed, such as elevation of the NaCl conten t, Iwadd ition of bile salts
derivatives,"' o r alkyl benzene sulpho nate. lo Significant variation in selectivity can be
observed when the several brand s of TCB S aga r are com pared.
Other media have also been devised which purport to be selective. Several plating
media utilize sucrose fermentation as a differential characteristic.
For
example brom
thymol blue (BTB) Teepol agar of Akiyama et al.93 ncludes Teepol for selection and
sucrose ferm entation (via the b rom thymol blue-thymol blue indicator) for differentia-
t ion. Sakazaki" modified BTB Teepol by sub sti tuting sod ium heptadecyl sulpha te
(Tergitol
7)
for Teepol and found i t to be m or e selective. Tee pol ca n also be replaced by
lauryl sulphate.99 Polymyxin-tylosin sucrose salt (P T S S) a ga r can provide differentiation
of Vibrio spp. on the basis of sucrose ferm entation , util izing antibiotics, rather tha n
detergent, fo r selectivity.62
W ater blue, alizarin yellow ag ar (WA)"' incor pora tes water blue and alizarin yellow in a
medium suitable fo r distinguishing between V. parahuemolyticus and
V .
alginolyricus.In
addition to dyes, WA contains beef extract , peptone, sodium chloride, Teepol, and
sucrose. Fermentation
of
sucrose by
V.
alginolyticus,
when it is gro w n on WA , results in a
reduction in the pH of the medium, thereby cau sing the me dium t o assume a blue hue,
caused by a chan ge in the dye, water blue, induced by the altered pH . Alkalinization of
the medium by sucrose negative strains of V. parahaemolyricus results in an orange-
yellow color, caused by the pH effect on the alizarin yellow dye.
A fermentable c arboh ydra te other than sucrose, such a s arabinose, was employed by
Horie et al.," ' who devised an arabinose am m on ium sulfate cho late (AAC) medium
which contains sodium c holate at a pH of 8.6. Un fortuna tely, there is a tendency for the
medium to provide an underestimate of the true incidence of
V.
parahaemolyticus
because arabin ose ferm entation is a variable chara cterist ic of the species (see Table 2).
So diu m cholate was used by Watkins et al."' to inh ibit the grow th of Gram-posit ive
organisms on the prim ary isolation medium. Co pp er su lfate has been used to inhibit
V
alginolyticus, a species closely related t o V. parahuemolyticus, with galactose included as
a ferm entab le carbohydrate." '
A nonselective medium was devised by B aross an d L i~ to n "~ * " ' mploying starch
hydrolysis as a differential Characteristic. The Baross and Liston medium contains no
selective agents and the pH of the medium
is
adjus ted to
7.5.
Inoculated plates are
incubated in an anaerobic jar for 36 to 48 hr a t 37 C.
For
samples conta ining large
numb ers of Bacillus spp., penicillin (20 U/mP) is added to achieve selection of Vibrio
SPP-
Twedt and Novelli 'I8 carried out a systematic study
of
media a nd media consti tuents
proposed for isolation of V. parahaemolyricus. Penicil lin in co 6o ra ted into a medium
of
'alkaline pH proved to be more useful than
a
variety of selective agents that had been
suggested by investigators fo r V. parahaemolyticus. Th ese included potassium tellurite,
sod ium deoxycholate, Teepol, and 6% NaC l, all of which were considered t o
be
useful in
the isolation of
V. parahaemolyticus.
With starch hydrolysis as the differentiating
characteristic, the final formulation included pep tone
(2'39,
yeast extract
(0.2%),
co rn
starch (0.5%), Na Cl (3.0% ),-@nicillin (2 U/mP ) , and ag ar (1.5%), pH 8.0. Subsequently,
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Vanderzant and Nickelsonw modified the medium of Twedt a nd Novelli lightly, using
7% NaCl and
1
corn starch, finding results superior to those from other media
recommended for recovery of
V.
parahuemolyticus.
Because of the high cost and great difficulty in obtaining TCBS agar in India,
investigators there have devised a less expensive and simpler plating media. De et
described
VP
agar, similar to TC BS a nd containing sod ium taurocholate and sodium
lauryl sulphate. Sucrose Teepol tellurite (SIT) agar, described by Chatterjee et a1.,*
contains peptone, beef extract, Teepol, potassium tellurite, sucrose, bromthym ol blue,
and agar. In the U.S. CB S is not difficult to obtain but n ot all laboratories, especially
clinical laboratories, routinely stock this medium. So me workers have reported
satisfactory results using more com mon media, such
as
mannitol salt agar and xylose
lysine deoxych olate (XLD ) agar amended with salt an d starch, for isolation of
V
parahaemolyticus.
In addition, well-trained and observant technical staff have noted
aberrant biochemical and morphological characteristics of cultures grown on conven-
tional bacteriological media and, in their follow-up, detected the presence of V.
parahuemolyticus.
C. Recovery of
Stressed
Cells
Prob lem s of heat and cold stress on V.parahaemolyticushav e been examined in detail
by food micr~biologis ts .~*-~his aspect of the biology of V parahaemolyticus is
interes ting since seafood is preserved by chilling
or
freezing and m ost seafood is cooked
well, or a t least heated, before consump tion. V.parahaemolyticur iswidely recognized a s
being sensitive to cold when it is present in fo od an d water.27 - In add ition, effects of
heat a nd cold stress of
V.
arahaernolyticus
are altered significantly by the m edium and
composition of the diluent to which they are exposed during and after tress.^'**^^'-^^^
Va nde rzan t et al. evaluated several proc edur es fo r isolation of V.parahaemolyticus
which were applied t o recovery of stressed cells. They co ncluded that TS B containing 7%
NaCI, when used with modified agar of Twedt and N ~ v e l l i , ~as more effective, but less
selective, tha n G STB and TC BS agar. Beuchat reported t ha t TSB to which
7
NaCl
had been add ed a nd GST B were inferior to arabinose ethyl violet broth9 an d to w ater
blue-alizarin yellow broth for enrichment and efficiency of recovery of cold- and heat-
stressed V. parahaemolyticus.
D.
Characterization
of
Isolates
Pure cultures obtained using any of the above methods can be characterized and
identified using a scheme similar to those m entioned ab ove. B iochemical prop erties of V.
parahaemolyticus can be determined using conventional media supplemented with N aCl
at approximately
1
to 3% final concentration. While V arahaemolyricus grows well on
ordinary blood agars and on Mueller-Hinton aga r, as well as most media containing
NaCI, there are particular media, e.g., M R -V P an d decarbox ylase media, to which NaCl
must be added in approximately 2%
concentration fo r significant growth to occur.135
A
specific medium, Wagatsuma medium, is employed
to
test fo r hemolysis of blood. The
reaction observed on Wagatsuma medium is termed the Kanagawa phenomenon
(KP).6736his medium contains: yeast extra ct, 5 g/P; peptone, 10g/P; mannitol, 5 g / P ;
K2HP0 , ,
0.5
gin ; NaCI, 70 g /Q ; agar,
15
g/ P; an d c rystal violet,
1
m Po f a O.l%solution.
Freshly dra wn and washed hum an blood cells are add ed to the cooled, prepared medium.
Results of the hemolysis test are not valid if the medium is held longer than
24
hr af ter
i n o c ~ l a t i o n . ~ * ~ ~new method for testing the Kanagawa phenomenon, using a liquid
medium, was reported by Ohashi et a]. Chun et al.8 reported that variable
KP
reactions will occur on W agatsuma m edium, depend ing u pon th e presence or absence of
fermentable carbohydrates in the medium.
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E. Enumeration of V.pmoliaemolyticus
The two most widely used me thods for enum eration of
V.
arahaemolyticus in food o r
water samples are the most probable number (M PN ) and the mem brane f il tration ( M F)
methods. Direct plating, rath er than M F, can be used if the num ber of cells in thesam ple
are high enough. M PN me thods uti lize enrichment broths, such a s those discussed vide
supra). Replications of three or five tubes can be used, with inoc ulation of a t least three
different sample volumes. A n exam ple of the M P N would include a series
of
lo-, 1 and
0.1-mP volumes of sample inoculated into three tubes of broth for each volume or
dilution employed. S am ple volumes larger than 10 m P can be concentrated using a
filtering system fitted with a 0.45-pm membrane filter. The filter with trapped cells,
can be placed into enrich me nt broth tubes, and the proc edur e carried out in replicate.
lsolation media, which a re often replicate plates conta ining any of several acceptable
media, are inoculated from tubes showing growth. These are incubated
as
for the
nonquan titative isolation. Nearly all enrichme nt bro ths and plating media described fo r
V. parahaemolyticus
have been used in th e M P N proc edure by investigators whose work
has been cited herein. At the present time, no universally accepted, standard method
exists for the enumeration of V.
parahaemolyticus,
i.e., there is none which is the
equivalent of the test for acid and g a s formation in lactose broth and presumptive,
confirmed, a nd completed tests employed f or enum eration of coliforms. Thus, the
inoculated plates are incubated and colonies must be picked for purification and
characterization. T he exten t
of
the testing proc edur e employed in the characterization of
strains, as well a s the "presumptive"
or
"confirmed" appellations remain subjective. These
are determined by the govern me ntal agency, individual labo rato ry,
or
the investigator
conduc ting the study. H owever, H ugh and Sakazak?' have published a list of minimal
characters suitable for the identification of
V .
parahaemolyticus and this has proved
effective in many instances (Table 2).
A
multitest, presump tive identification med ium specific fo r
V. parahaemolyticus
has
been developed and it offers a relatively quick and inexpensive aid in biochemical
characterization an d screening of large nu mb ers of isolates suspected to be
V .
parah~ernolyticus.~~his med ium, prepa red in tubes, gives a cha rac teris tic overall
reaction for
V. parahaemolyticus,
derived from arginine dihydrolase (-), manni tol
fermentation
(+),
sucrose and lactose fermentation (-), HIS product ion
(-),
gas
production
(-),
and indole product ion (+).
The membrane filtration technique, which has been successfully employed to
enumerate oth er tax ono mi c groups of bacteria, was first devised for vibrios by Horie et
al.
'
numeration of
V. arahaemolyticus
can be effected w ith a n arabinose, amm onium
sulfate, sodium cholate (AAC) medium at pH 8.6. After fi l tration of the sample, the
membrane is placed on the A AC medium an d incubated a t 42OC. Gr ow th at 42C has
proven to be an important differential characterist ic
of
V. parahaemolyticus. Yellow
colonies appe aring on the fi lter, incubated on the surface of the AA C medium. a re
arabin ose fermenters, an d therefore ar e presumed to be
K
parahaemolyticus.
Unfortunately, a s noted abov e, there is a tendency for und erestim ation of the true
incidence of V. parahuemolyticus, when this method and medium are used, because
arabin ose ferme ntation is a variable characteristic of th e species.
Many of the agar plating media discussed herein have been adapted for use with
membrane f i lters, and TC BS ag ar is one that is most com monly employed. W atkins et
al."' reported
a
me thod involving m em brane fi l tration w hich was specifically designed
for enumeration of V. parahuemolyticus. The primary isolation medium fo r the method
was based on the ab ility
of V .parahaemolyticus
to grow in the presence of
3%
NaCI, a t
high p H , i.e., 8.6, and a t 41OC. S od ium cho late in the medium served to inh ibit grow th of
Gram-posit ive organisms. G alactose provided the source of carb ohy dra te
for
V.
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I 89
parahoemolyticus.
V.
alginolyricus, a closely related species, was inhibited by copper
sulfate. Attempts were made to achieve rapid identification of V. parahoemolyticus,
without transfer of each individual colony to other test media.
For
example, the
membrane filters, on which the colonies were growing, were transferred successively to
galactose a nd sucrose fermentation media, with the oxidase test performed as the final
step. Th e procedure a s described by the auth ors can be done within
30
hr, yielding 95%
accuracy of identification.
IV.ECOLOGY OF V . P A R A H A E M O L Y T I C U S
A. Geographical Distribution
V
parahuemolyticus is, indeed, widespread in occurrence. First recognized in
japan,
.95.139
it has also been isolated from samples collected
in
Korea,Ia Thailand.Io4
Indonesia,47 Vietnam,14' China,'42 India,'43-146ran,147R u s ~ i a , ' ~ 'nd A ~ s t r a l i a . ' ~ ~ * ' ~
The occurrence of
V.
parahaemolyricus in Europe has been reviewed by Leistner and
Hechelmann,'" and cou ntries of isolation include the
nether land^,^^.^^""
Great
Britain,63"s2 Denm ark,
102,153
Germany,'5'' 'l '4 Italy,'9*'5sScotland,6' Spain,'" and the
Black, Baltic, No rth, and M editerra nea n Seas."' Re ports fro m Africa include isolation
of V . parahoemolyticus in Togo'56 n d M a d a g a ~ c a r . " ~n the Western Hemisphere,
V.
parahuemolyticus has been isolated in
In the U.S. V. parahuemolyticus was reported by W ard'60 o n the basis of serological
relatedness of V. arahaemolyricus-like organisms in sediment samples collected from
the Southeastern coast. S ince then a r epo rt of the occurrence of this species from nearly
every coastal state has been m ade, including New Ham pshire,94Massachusetts,I6' Rhode
Island,I6' Maryland,97.'63-'64Virginia, '63 North C a r~ l i n a , ' ~ ' outh Carolina, '66 Flor-
ida,'67 th e Gulf C o a ~ t , ' ~ ' - ' ~ ~regon,I7' W a ~ h i n g t o n , " ~ " ~aw aii,m a nd A 1 a ~ k a . I ~ ~
V. parahuemolyticus can be isolated throughout the estuarine environment. Water,
sedimen t, suspende d particula tes, plank ton, fish, and shellfish samples have been shown
to harbor the organism. The principal features which influence the ecology of
V.
parahuemolyticus are salinity, seasonality, and association with higher organisms.
V.
parahuemolyticus is most com monly an inh abit ant of estuaries and is infrequently fou nd
in freshwater or full-strength seawater. The seasonal cycle
is
temperature dependent,
with higher numbers evident during warmer summer months.
V.
parahaemolyricus is
associated with a number of higher organisms, notably plankton and shellfish.
Panama, '59and the U.S.
B. Seasonal Variation
In most of the geographical areas where V. parahaemolyricus
s
known to occur, the
incidence of the organism s follows a distinct seasona l cycle, with highest counts recorded
in the summ er and fall and lowest coun ts in the winter. This phen om enon w as first noted
in Jap an by M iyamoto et al. '39 and has been confirmed by a number of other Japanese
investigators including Nishio et a1.173v174nd Sh in et al.175 n other countries, the
seasonality of the organisms subsequently was recorded, including
Australia,'50 and the
U.S. 97,116,117,1W176
In te re stin gly , Th o m ps on a nd V a n d e r ~ a n t ' ~ ~
reported that a seasonal cycle for
V.
parahuemolyticus
could no t b t detected in the Gulf
of Mexico, but noted that temperatures were higher year round than in other
environments studied, with the lowest temperature only 11.6OC. In the Chesapeake Bay
and other areas, the organism is absent fro m the water column during the winter mon ths,
but can be isolated from sediment throu gh out the inter.^"'^ In other environm ents it
remains
in
the water column, but at greatly reduced numbers.63
In tropical countries, the s easo nal cycle of V.parahaemolyricus s correlated with rainy
and dry seasons. In Vietnam, highest numbers a re found in rainy months (M arc h and
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April) and lowest numbers are found
in
the dry season (December to February). '"
Curiously, the opposite was found in To go (West A frica) and in Indonesia where highest
cou nts were found a t the end of the dry season (April) and the lowest in the rainy season
( J ~ n e ) . ~ ~ . " ~alinity measurements, not recorded during the Vietnam
or
Indonesian
study, are available for Togo, where salinity was found to be highest during the dry
season, i.e., greater than 12% per thousand, when counts of
V.
parahaemolyticus also
were highest. D urin g the rainy season, a salinity of 1.6 t o 4.2% per thousand was
recorded, which is less tha n o ptim um fo r V. arahaemolyricus. At th at time, few isolates
were recovered.'56
Besides tem per atu re and salinity, seasonal va riatio n of V. parahaemolyticus can also
be influenced by interac tions with plank ton a nd higher organisms. K anek o and
~ ~ l ~ ~ 1 1 9 7 . 1 6 4 . 1 7 8tudied the season al cycle of
V .
parahaemolyticus and zooplankton in
Chesapeake Bay and reported that from late spring to early summer, vibrios over-
wintering in sediment enter the water column by atta chm ent to zoop lankto n. Interaction
between sediment, water, an d zoo plank ton was found to be essential . As temperatures
increased and vibrios proliferated, V. parahuemolyticus was readily isolated from the
water co lum n. M iyam oto and K ~ r o d a ' ~ ~uggest that a
Bdellovibrio
lethal
for V.
parahaemolyticus may also play a role in the seasonal cycle
of
the host. These
investigators found that Bdellovibrio can lyse
V .
parahaemolyticus a t temperatures as
low as 5OC, but not at higher temperatures, i.e.,
35OC.
Thus, at lower temperatures,
during fall and winter months when Bdellovibrio can actively lyse the host, V .
parahaemolyticus does not readily proliferate. Obviously, the seasonal cycle of V.
parahaemolyticus
may be influenced by a va riety
of
factors a nd a com plex interaction of
these, especially tem peratu re, salinity, ad sorption , a ttach me nt, plank ton, parasites, etc.
C. Correlation
with
Environmental Parameters
Th e correlation
of V. parahaemolyticus
and its Occurrence in the environment with
indices of pollution
is
not at all clear. Several
investigator^""'^^'^^
reported greater
concentrations
of
V.parahaemolyricus in polluted wa ters vs. nonp ollu ted waters. On the
o th er ha nd , T h om p so n a nd V a n d e r ~ a n t , ' ~ ~utton,15' Ka neko a nd C 0lwe11,~' nd Jon as
et a1.'65 re po rt no significant correla tion with cou nts of V. arahuemolyti& an d pollu-
tion indices, such as total cou nts of coliforms, fecal coliforms,
or
Escherichia coli. These
variances can be resolved if other factors associated with pollution ar e considered, such
as the concentration of nutrients and suspended particulates, rather than coliform
counts. Coliform indices usually correlate well only with occurrence of allochthonus
bacterial pathogens, such as Salmonella spp. and not wi th the autochthonous,
potentially pathogenic bacteria, such as V parahaemolyticus.'".'8'
Watkins a nd Cabelli'62 reported tha t ads orp tion of V. parahaemolyticus to
particulates is greater in water of lower salinity and that the numbers of V.
parahaemolyticus
may be indirectly related to pollution because of co ncu rren t inp ut of
particulates. Otherw ise there is no direct corr elat ion with pollution. In studies carried ou t
in Chesapeake Bay, the occurrence of
V.
parahaemolyticus did show a positive
correlation with coliforms in the area of the Chesapeake Bay surrounding Baltimore
Ha rbor, but was not a s highly correlated
as
was Sa l m one l l u sp p .~ohe coliform count.'"
In a subsequent survey covering the entire Chesapeake Bay, multivariate regression
analysis
of
the da ta revealed that salinity an d dissolved o xyge n conc entration were most
closely correlated with the incidence of
V. parahuemolyticus.
The frequency of
occurrence, i.e., tota l nu mb er of
V.
parahaemolyticus, increased with increasing salinity
and decreasing dissolved oxygen (DO) concentration, the latter most likely reflecting
increased n utrient concen tration in eutrop hic area s of the Bay.'83
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V. parahuemolyticus has been isolated from freshwater in several areas. Well water
from Indonesia was found positive in several instances where samples from Ja va w ere
e ~ a m i n e d . ~ 'n Calcutta, India, V.
parahaemolyticus
has been isolated from w ater sam -
ples and fish taken from essentially fresh water in the River Hooghly, approximately
50 miles upriver from the Bay of Bengal.145
V. parahuemolyticus
is widely distributed in
this ar ea , being found in
40
of pond wa ter sample s "having p ractically no sa1inity"and
fed chiefly by rain water.lE4 In the Ch esap eak e B ay, V. parahuemolyticus has been
isolated fr om the U pper Bay, as well as the uppermost reaches of the James River and the
Po tom ac River."' Sayler et al."' examined the Uppe r Chesa peake Bay an d recovered
several Isolates from samples in low salinity areas. An isolate was obtained from
suspended sediment where the water temperature was 4.3OC and the salinity was below
the detectible limits of the salinometer. Tidal transport of
V.
parahaemolyticus, no
do ub t, plays an imp orta nt role in the occurrence of the organism up-river of estuaries.
Ayres an d reported higher concentrations of V. parahaemolyticus in muddy
sediment than
in
sand or gravel sediment, indicating the influence of organic matter on
Occurrence an d survival of this species. The transfer of vibrios fro m sediment
to
the water
column and from the water column to marine animals was examined for "V. parahae-
molyticus biotype 2 . i.e., V. alginolyticus. by G auth ier and Clement.'86 These
investigators reported that transfer
of
vibrios via sediment was very important in
persistence in the water column and marine anim als and th at colonization of water from
sediments was not observed a t temperatures less tha n 16OC. As noted ab ov e, ads orp tion
of V. parahaemolyticus to particulates is greater in lower salinity waters,16' a finding
consistent with observations made in Ch esap eake Bay'" and in Ca lcutta.
Thus,
the
Occurrence of V.
parahaemolyricus
in brackish
or
freshwater environments can be
concluded to be significantly affected by the occurrence of particulate matter, nutrient
conc entration , and salinity.
D. Occurrence
in
the
Open
Sea
V.parahaemolyricus
has been isolated rarely from pelagic regions of the world ocea ns.
I t appears to be limited to inshore coastal an d estuarine areas. Aoki et al.142eported the
isolation of V. parahaemolyricus from the op en sea of Japa n, but neither Horie et al .9s9i1 4
nor Miyamoto et al.I3' found
V. parahaemolyticus
in water samples collected from
pelagic areas around Japan. The inability to isolate
V parahuemolyficus
from pelagic
areas was also reported by V arga an d H irtle"* in Ca na da an d Bockemuhl and Triemerls6
off the co as t of Africa. Baross and Liston"' noted th at the incidence of V .
parahaemolyticus in seawater decreased with dep th off the W ashington co ast and very
low num bers w ere foun d in deeper sedime nts. K anek o an d Colwe11''' collected samples
along fo ur transects on the con tinental shelf off the southeastern U.S. and did not isolate
classical
V.
parahaemolyricus from any of the water, sediment,
or
plankton samples.
However, a num ber of vibrios very similar to V. parahaemolyticus was recovered. Th e
absence of
V. parahaemolyticus
from the open oce an is most likely a result of low water
temperature, high salinity, an d low nutrient c onc ent ratio n, since V.parahaemolyricus
unlike other marine bacteria, is cold sensitive and does not survive well in low nutrient
waters. Another major factor to
be
considered when examining th? incidence of
V .
parahaemolyticus in the open ocean is hydrostatic pressure, the effect of which was
studied by Schw artz and Colwell,"' who reported th at
V parahaemolyticus
was unable
to
grow at a ny hydrostatic pressure simulating the de ep ocean env ironment, i.e., from 200
to
1000
atm of pressure. Thus, he inability of V. parahaemolyticus to tolera te elevated
hydrostatic pressure supp orts the conclusion tha t neritic o r estuarine waters are habitats
of V. parahaemolyticus.
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E. Association with Higher Organisms
V .pnrahoemolyricus is now known t o be associated with a variety of higher organisms
living in &hemarine an d estu arin e environm ent. including plan kto n, fish, an d shellfish.
Shellfish. in particular, a re im por tan t because of the possibility of hum an disease arising
from their ingestion. Fishbein et repo rted the isolation of V.parahaemolyt icusfrom
30 different marine species, including clams, oysters, lobsters, scallops, sardines, shr im p,
squid, crab, eel, and various species of fish. From 1969 to 1972, 546 ou t of 635 (86%)
seafood samples examined by the Food and Drug Administration were found to be
positive fo r V. p a r a h a e m ~ l y t i c u s , ' ~ ~erh aps a biased result because man y of the samples
were collected d urin g outbre ak s of enteric disease. N evertheless, oth er equally extensive
studies have been reported which document the presence of V. parahuemolyticus in
Coun ts o f
V.
parahaemolyticus
lams, mussels, and oysters.
in oysters c a n be as high a s 1300per g ram of tissue.'70 Mo re typically, the n um ber s ar e
10
per gram. Isolation of
V.
parahuemolyticus from c rabs has been rep orted by Fishbein et
al ,,la9Krantz et al.,lw Barrow an d Miller,'s2 Colwell et aI.,I9' and D e et al.,14' an d from
shrimp by Vand erzant e t a1.,1689192ishbe in e t a].,*' De et al.,145 elsenfeld a nd Cabirac,I7'
and Joseph et Co ncen trations of V. parahaernolyricus in crab s can be as high as
10'
per gram of meat.
V.
parahuemolyticus
has not been isolated from fish as frequently or a s readily as from
filter feeding
invertebrate^. ^^ ^
Nevertheless, a wide variety of marine and freshwater
fish has been shown to harbor the
o r g ~ n i s m . 6 * ' 9 9 ' ' 4 ~ 4 6 ~ 1 7 0 ~ 1 9 3 - ' 9 ~
In addition to the com mensal, or symbiotic, association of
V
parahuemolyticus with
higher organisms, a pathogenic relationship is possible also . K ra nt r et isolated
V.
parahaernolyiicus
from lethargic and mo ribund crabs, and Brinkley et al .I9' and Tubiash
et al.IP6 reported an association of
V.
parahoemolyticus with disease in lobsters and
bivalve mollusks, respectively. Vanderzant et a1.16a,192n d V anderzant a nd N i c k e l ~ o n ' ~ ~
reported the death of shrim p in ma ricu lture caused by infection with V. parahaemolyri-
cus. An outbre ak of disease in a Mexican shrim p hatchery has also been shown to be
associated with
V .
parahaemolyricus
(D.
Danald, personal comm unicat ion). Pathogenic
properties of V parahuemolyticus may be a s important in the maricul ture of shr imp
or
other invertebrates, as V. anguillarum has proved to be in fish hatcheries.
62 . 94 . 99 . 106 , 107 , 117 . 149 , l50 . l56 , l~7 , l77
F. Kanngawa Phenomeno n Positive Y.pwuhernolyticus
in
the Environment
An important, as yet, unanswered question concerning
V
parahuemolyticur
is
the
exact relationship between the Kanagawa phenomenon (KP), pathogenicity of the
organism, and the enviro nm ental reservoir of the organism. The re
is
a significant
correlation between the K P and pathogenicity of
V
parahuemolyticus, bu t the reason is
not yet known. S akaz aki et al .19 7 eported that 96.5% (2655 out of 2720) of strains
isolated from human patients was K P + while only
1% (7
ou t of 650) of isolates fro m the
environment was KP+. Oth er investigators have reported similar findings. Thom pson
and Vanderzant"' foun d only
4
KP+ st rains out of 2218 total isolated fro m water and
sediment
in
Galveston Bay. Sp ite et a1.'99foundone isolate in an oy stcr from U.S. waters.
Three strains o ut
of 251
water, fish, and shellfish stra ins exam ined in were KP+.
Ayres and Barrow63found no
KP+
strains ou t of
1484
isolates obtain ed from Brit ish
coas ta l waters . Lei s tner and H~chelman '~ 'ound only
1
KP? strain ou t of 1708 f rom
European waters. Su tton'so concluded that
2%(22
ou t of
986) of
the strains isolated fr om
oysters growing in Australia was
KP+.
An ecological study of K P+ strains fou nd in
Ja pa n was reported by Wagatsuma.2w who recovered KP + strains from sam ples of mu d,
seawater, and oysters. H owever, isolation of K P+ strains was rare in the absence offo od
poisoning
in
a nearby co mm unit y, whereas K P- strains were always isolated, regardless
of docu me ntation of food poisoning cases in the com mu nity.
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These findings are best explained perha ps by a process of natura l selection of
K P +
strains
in the intestine and better survival of KP- strains in the environment. S akazaki a nd co-
workers dem onstrated tha t K P+ vibrios do, in fact, multiply more rapidly tha n
KP-
strains in ligated rabbit loops and postulated, therefore, that selective multiplication of
KP+ organisms occurred in the intestine, even
i f
KP- strains were pred om inant in the
ingested food sample. Conversely, KP- strains survived longer in seaw ater tha n KP+
strains and grew better at 25OC than the
KP+
strains. Interestingly,
KP+
strains grow
better at 37C and under acid conditions.
Thus, the phenomenon
of
KP+ V.parahaemolyricus isolation almost exclusively from
hum an patients and KP- strains from seaw ater and seafood may be only a result of
selection and environmental pressure, with selection occurring in the intestine for
K P+
strains and in the m arine environm ent for
KP-
strains, a provocative ph enom enon yet to
be elucidated.
V.
RELATIONSHIP O F
V.
P A R A H A E M O L
YTICUS
WITH
B A C TE R IO P H A G E A N D BDELLOVIBRZO
Bacterioph ages specific fo r
V.parahaemolyticus
were isolated from fecal and seaw ater
samples in Japan by Nakanish i et a ~ ~ klarow et exam ined Atlantic coastal
sediment and recovered a bacteriophage specific for
V. parahaemolyticus
which was
morphologically sim ilar to phage isolated fro m W ashington c oastal waters.M Baross, et
al.17~206307ecovered bacteriophages active against V.parahaemolyricus from 177 to 643
samples of marine molluscan shellfish, crustaceans, seawater, and sediment collected
from W ashington and Oregon waters. Titers of
V.
parahaemolyticus
bacteriophage were
fou nd to increase with increasing water temp erature du ring the warm months of t.he year.
The titer of b acteriophage was discovered to be pr opo rtion al to the increase in the
numbers of mesophilic vibrios, but not with the incidence of V. parahaemolyticus.
Lysogenic bacteriophage could be induced from a n agar dig es tin g vibrio and it was
speculated tha t bacteriophage may, in fact, be impo rtan t in explaining the variability of
marine vibrios, with respect to phenotypic characteristics. and possibly even in animal
and human pa th~ ge nic i ty . ~ nterestingly, no bacteriophage act ive against V.
alginolyticus has been reported, even though V.alginolyticus occurs in larger num bers in
the marine environment than does
V. p a r a h a e m o f y r i ~ u s . ~
Marine
Bdellovibrio
capable of lysing
V.
parahaemolyticus
have been isolated from
Chesap eake Bay and Osaka Bay.79.20*-210solates of marine
Bdellovibrio
from both
U.S.
and J apa nes e waters are reported to have a broad host range, an d a t least in Chesa peake
Bay, demonstrate a seasonal cycle similar to th at of the host.2
VI.
PATHOGENICITY
A. General
Diarrheal disease caused by K parahaemolyricus is a food-borne infection related
chiefly to the ingestion of seafoods. The organism is autochthonous in waters of low
salinity, e.g., estuaries, and ap pea rs to have a n essential role in the ec olo gy of coastal
marine
environment^.^^
Its involvement as a pathogen f or m an, there fore, is usually
inadvertent through contact with contaminated and improperly handled seafoods. V.
parahaemolyricus is not regarded a s an acutely infectious organism, althoug h there is
little doubt that it is capable of causing serious illness. Results of experiments in which
human volunteer subjects were fed broth cul tures of clinical i ~ o l a t e s ~ ~ ~nd a n accident
in which a labora tory worker ingested approx imate ly
lo5
viable cells97make i t quite
clear that V .parahaemolyticus, occurring in sufficiently large num bers, can cause ac ute
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94 C R C Crirical Reviews in M icrobiology
gastroenteritis in man. Sanyal and Se nz i3 onfi rmed the enteropathogenic properties of
KP + strains in hum an volunteer experiments. S ignificantly, sym ptom s of gastroenteritis
appeared rapidly in individuals who had ingested at least
2
X
10' to
3
X
lo7
CFU
of KP+
V.
arahaernolyticus. Volunteers, receiving KP-
V.
parahaemolyricus in concentrations
ranging from
4
X lo9 o 1.6X 10 C F U , did not have diarrh eal symptoms. Earlier studies
had shown also tha t inge stion of lo 9 cells of
KP-
strains by human volunteers had
seemingly no ability to induce dia rrh eal Th us far , there is no indication,
from either epidemic
or
sporadic cases, that e nte rop ath oge nici ty is associated with
particular serotypes.*" A11 serotypes have been isolated from humans, seawater, and
seafishes. Lo ngitudinal studies in the same localities reveal th at p artic ula r serotypes d o
not predom inate in hu ma n il lness and marine enviro nm ents fro m
1
year to the next.
Serotypes frequently isolated from seawater and seafish during shorter periods tend to
predominate in cultures from patients.'"
Th e nature of the illness caused by
V. arahaernolyricus
leads one to conclude that a n
exoto xin, i .e., entero toxin, should
be
the responsible. identifiable substance. Interest-
ingly, feeding experiments showed that only administration of live cells produced
intestinal tract-related sym ptom atolog y, belying the possibility of preform ed exo toxin in-
gestion with incriminated foods. F ur the r evidence fo r infection lies in the fact tha t l o* o
10I2organisms are required to establish disease, an d in the acute stages,
lo6
o 10' organisms
per milliliter of feces can be
I t
is not yet known whether the pathological
effects of the organism ar e due to toxin production, direct dam ag e to the intestinal tract
fro m microbial invasion, both, or neither. The ability to multiply in the intestinal tract is
generally associated with the K P reactivity of the vibrio, which co m mo nly is considered
to
be
the essential index
of
capacity for en teropa thoge nicity in hum ans. This hemolytic
characteristic, described in gre ate r detail below, is fou nd in appr oxi ma tely 95% of clinica l
isolates and is rarely associated with en viro nm ent al isolates. H um an volunteer and
ani ma l experiments generally tend t o subs tant iate this view, altho ugh the significance of
the few cases caused by KP- stra ins remains partially unexp lained. T he suspicion is that
these strains produce hemolysin but in very small quantities. This has been bo rne o ut, in
fact, by using a serological meth od
of
detection, w hich reveals that, indee d, hemolysin is
produced by some presumed KP- strains.13' Th e suggestion has been m ad e that V.
parahuemolyricus produces a n cnterotoxin.'I6 Th e possibility th at enter oto xin is
produced and contributes to the disease process to some extent, regardless of KP
reaction, has yet
to
be clearly es tabl i~hed. ' '~
B.
Toxin
Assessment
1 . Mouse Inocularion
Stu die s using inoculation in adu lt and suckling mice generally confirm th at large
num bers of vibrios are required to cause lethality. Fujino* observed the lethal toxicity of
V. parahaemolyricus
to m ice an d guinea pigs. Zen-Yoji e t al."' used mouse foot pad
measurements to show that KP+ and
KP-
strains were uniformly toxic and caused
highly edematous reactions. In contrast, V. alginolyticus strains were relatively
benign, producing significantly less swelling. Co nc ent rate d cell-free filtrates introduced
via foot inoculation showed lethal toxicity, whereas all lysates provoked only slightly
edem atous responses. A ttempts to detect a n E. coli-like heat-stable toxin (ST) in suckling
mice were inconclusive. Recently, Jo hn so n and Calia,'" using co nce ntra ted filtrates,
demonstrated weak ST-like responses in the same system.
W hole cells of
V.parahaernolyricus
administered per
os,
SC, r I P resulted in mouse
lethality caused by septicemia.' Sa ka zak i et al." fo un d th at 0.5 mP f subgroups I and 2,
now
V.
arahaemolyricus and
.V.
lginolyricus, injected IP int o mice resulted in lethality
within 24 t o 48 hr. Lethality is dose dep ende nt. Tw o differen t KP + clinical strains
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inoculated into NMRI mice yielded LDso values a t concentrations 2 10' but were
nonle thal a t lesser concentrations.'" Inoc ulation s of cell-free filtrates
of
over 200
separate clinical isolates, even at IOX concentration, did not cause death of
NMRI
mice,221 urther emphasizing the a pp are nt requirem ent f or large num bers of vibrios to
cause significant disease.
2. Rabbit Inoculation
The rabbit ligated ileal loop (RIL)model has been used by several groups of
investigators to describe the enteropathogenic character of V. parahaemolyticus.201*
Whole cultures of V. parahaemolyticus and V. alginolyticus introduced into RIL can
elicit "enteritis" reactions"." Both KP-
and
KP+
strains of
V.
parahaemolyticus may
cause dilatation in RIL.Generally,
KP+
strains are positive m ore frequen tly in the RIL
system, than are
KP-
strains.2'4225~z2*ell filtrates are not capable of eliciting a fluid
accumulation in
RIL,
lthough based on early studies, Sakazaki et al.'lS suggested that
an enterotoxic substance was produced by V. parahaemolyricus and played a role in
inducing gastroenteritis. This appeared t o be substantiated when hea ting at 100C for 30
min eliminated activity of tenfold co ncen trated cell-free filtrates. R ecently, Jo hn so n and
Calia233 ave shown t hat concen tration of filtrates produces solutions containing >20
NaCI and reported tha t media containing N aC l2 4 95 induced positive responses in
RIL.
They concluded that cell-free filtrates of
V.
parahaemolyticus, even if concentrated
tenfold, after dialysis, were not capable of causing dilatation in
RIL,
when NaCl
concentrations were
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KP- strains, the effect
w a s
more rapid a nd complete with the KP- strains. It was
subsequently noted that
V.
parahaemolyticus adhered rapidly to suspension-grown
HeLa cells and to human fetal intestinal cells (HFI ). KP -strains did not adhe re to HeLa
cells and adhered to H F I cells a t a much slower rate than did KP + strains. Adherence
appeared to depend upon interaction between the cell surface of the bacteria and the
epithelial cell, a carb ohy dra te on the ou ter m embran e of the bacterial cell wall apparently
necessary for the Using clinical str ain s of KP+ and KP- V.
parahaemolyticus, Sochard and Joseph were unable to differentiate between either
gro up similarly exposed to HeLa cells.2a Gingras and Ho wa rdz4 lso were unable to
detect significant differences in vitro, by radioassay. in the adherence of KP+ and KP-
strai ns of
V.
parahoemolyticus to human intestinal cells.
Culture filtrates of V. porahaemolyricus isolates from human gastrointestinal
infections, when exposed to Y-1 drena l cells, d o not cause morphologic change^.^^^-^@ In
contra st , H ond a et al .27 ave isolated a heat-labile fac tor from the culture fi l trates of
K P+ strains which causes morphological changes of Chinese ham ster ovary (C H O ) cells,
the premise being that this factor is an entero toxic substan ce separable from the cardio-
toxic hemolysin.
Oishi et al.2 45 ave recently described the presence of exohem agglutinins ( H A ) from
marine vibrio strains, which had tax ono mic properties similar to
V. parahaemolyticus.
HA showed remarkably high hemagglutinating titers for a wide spectrum of
erythrocytes. The more widely reactive H A were not easily inhibited, whereas the HA in
the g rou p with narrow er spectra, were specifically and co mpletely inhibited by L-fucose
an d D -arabinose. T he physiologic significance of HA is not clear, bu t they may eventually
emerge as an imp ortant feature of adherence, an essential step in pathogenicity.
Clinical exam ination of a few patients in India, Bangladesh,246 nd the U.S.,247who
were suffering from
V. parahaemolyricus
dysentery-like diarrhea, suggested that
invasion of the bowel may occur, i.e., blood a nd mucu s in the stoo l, polymorp honuclear
leukocytes
seen
by microscopy, and superficial ulceration of colonic mucosa seen by
sigmoidoscopy. These observations are significant when compared with similar,
experimental findings in
rabbit^.^^^'^^'
Boutin et a1.,232 sin ga direct fluorescent an tibo dy
method, studied invasiveness as a step in pathogenesis during host organ-bacterial
interaction. All strains tested, K P+ an d KP-, penetrated in to the lam ina pro pria of the
ileum and were eventually isolated f ro m the spleen. How ever, not all were ab le to cause
fluid dilatation
of
RIL.
KP- stra ins caused only occasional positive responses.
Interestingly. V. parahaemolyricus do es no t ex hibit a positive Se re ny test, i.e.,
penetration of the corneal epithelium
of
guinea Attempts to demon strate
invasiveness by V. parahaemolyricus an d oth er closely related vibrios with the He La cell
techniques described by Me hlman et reinforced the impression tha t, by this
experimen tal m ethod, a s well,
V.
parahaemolyticus penetrat ion
or
invasion of tissue is
still a questionable phenomenon.240
Joseph et aL2 found that a
KP-
strain injected into th e i leum of suckling rabb its
caused focal necrosis and appeared to adhere in damaged areas, but no t on apparen tly un-
effected tissue, leading t o speculatioh th at tissue dam age , adherence , an d invasion were
interrelated, possibly with toxicity. Fu rthe r examin ation showed that a dhere nce was
possibly mediated by lateral appen dage s (flagella), and played i n im po rtan t role in the
stimulation of fluid accumulation in R I L and lethality to mice by the whole, intact
Similar investigations reported simultaneously by Shinoda et al .2s
sub stan tiate d these results.
D.
Permeability Factor
Th e permeabili ty fac tor (P F ) or skin toxin, foun d to be capa ble of pro ducin g increased
vascular perm eability in the skin of exp eri